The long-term goal of this research is to elucidate molecular mechanisms involved in synaptic vesicles reformation after exocytosis. This is a function essential for sustained synaptic transmission. Understanding mechanisms underlying presynaptic function is important from many perspectives. Various motor and behavioral deficits may result from defects in molecules required for presynaptic functions; excess neurotransmitter release during epileptic seizures may result in excitotoxicity and consequent cell death in the brain; altered activity-dependent regulation of transmitter release may underlie deficits in learning and memory. This proposal exploits the convenience of Drosophila as an experimental organism for incisive experiments on the mechanisms of presynaptic function. Such studies in Drosophila are validated by numerous examples of conservation of neural mechanisms across phylogeny. The rate of progress of the proposed analyses in Drosophila is facilitated not only by its short generation time and facility for genetics, but also by novel resources provided by the Drosophila genome projects, and newly developed technologies for gene disruption, perturbation and replacement in vivo. The proposal seeks to use an integrated, multidisciplinary approach to analyzing mechanisms of synaptic vesicle recycling. Using the facility of Drosophila for transgene manipulation, the first specific aim this grant seeks to understand how novel presynaptic proteins, stonedA and stonedB, accomplish their essential roles in recycling synaptic vesicles. The second and third aims seek to identify novel proteins that function at the nerve terminal for recycling synaptic vesicles, by molecular, genetic, and cell biological analysis of two genetic mutants in Drosophila whose preliminary characterization indicates altered endocytosis at nerve terminals. The last aim of the grant addresses a fundamental question in the cell biology of neurotransmitter release: are there different pathways for transmitter release and synaptic-vesicle reformation? Combining transgene manipulation, genetic screens and molecular genetics, with novel and established cell biological assays for presynaptic functions in Drosophila, the proposed experiments, if successful, should contribute significantly to current knowledge of presynaptic functions.